Inhibition of membrane fusion proteins

ABSTRACT

Methods of inhibiting viral infection of a eukaryotic cell by a target virus having a class II virus fusion protein are provided. Also provided are methods of screening a test compound for the ability to inhibit infection by a virus having a class II viral fusion protein. Additionally provided herewith are aqueous-soluble proteins comprising a portion of a class II viral fusion protein comprising a Domain III of the viral fusion protein.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/678,467, filed May 6, 2005.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of GM52929awarded by the National Institutes of Health.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention generally relates to control of viral infection.More specifically, the invention is directed to control ofvirus-membrane fusion reactions of viruses having class II fusionproteins, and methods for identifying compounds that effect thatcontrol.

(2) Description of the Related Art

REFERENCES CITED

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The role of flaviviruses and alphaviruses in disease. The flavivirusgenus includes a number of serious human pathogens that are disseminatedin nature by mosquito or tick vectors (Lindenbach and Rice, 2001). Theflavivirus dengue virus is of particular concern as it has dramaticallyreemerged to become endemic in more than 100 countries including the US(Clarke, 2002; Gubler, 1998; Monath, 1994). Dengue is a major globalhealth problem and the estimates are that more than one-third of theworld's population lives in dengue fever endemic areas. The WHOestimates that there are ˜100 million cases of dengue infection and500,000 cases of the more lethal complication dengue hemorrhagic fever(DHF) per year, requiring 500,000 hospitalizations and causing ˜25,000deaths, mostly in children (WHO, 2002). This disease burden also has asignificant economic impact in developing countries. Dengue virus isclassified as a category A priority pathogen by the NIAID. Whilesignificant research on preventative vaccines is underway, it iscomplicated by the fact that antibodies can enhance infection leading toDHF and dengue shock syndrome (Rothman and Ennis, 1999). Control of themosquito vector is the current mode of prevention but is itselfproblematic and almost nonexistent in many endemic countries (Weaver andBarrett, 2004). Thus, alternative antiviral strategies are sorelyneeded. Other important flavivirus pathogens include Japaneseencephalitis (category B), the leading cause of viral encephalitis inAsia; tick-borne encephalitis virus (TBE) (category C), which causesthousands of cases of human illness yearly in northern and easternEurope; and West Nile virus (category B), which has emerged recently asa human pathogen in Europe and North America. Although there has been avaccine against yellow fever virus for many years, disease cases haveincreased to 200,000 per year with some 30,000 deaths, leading to theclassification of this flavivirus as a category C emerging infectiousdisease. The Flaviviridae family also includes the more distantlyrelated hepatitis C virus (HCV), an important blood-borne pathogenresponsible for severe chronic liver disease and liver cancer (Memon andMemon, 2002).

The alphavirus genus contains ˜24 virus species that are generallyspread in nature by mosquito vectors (Schlesinger and Schlesinger, 2001;Strauss and Strauss, 1994). The alphaviruses eastern equine encephalitis(EEE) virus and western equine encephalitis (WEE) virus cause severeencephalitis in humans. Venezuelan equine encephalitis (VEE) viruscauses encephalitis, myocarditis, pharyngitis, leukopenia, and hepatitisin humans, and leukopenia, encephalitis, and pancreatitis in horses(Griffin, 1986; Weaver et al., 2004). EEE virus is among the mostvirulent alphaviruses, with a case fatality rate of ˜35% in humans ofall age groups. Both EEE virus and WEE virus are endemic to the UnitedStates and responsible for periodic epidemics of encephalitis in humans(MMWR, 1992; MMWR, 1994). In the past, VEE was weaponized by both the USand the former USSR, and currently EEE, WEE, and VEE virus are allclassified as category B priority pathogens. Given the known spread ofmosquito vectors into new regions, these viruses are also potentialemerging pathogens (Weaver and Barrett, 2004). There are no effectivetherapeutic drugs and, similar to flaviviruses, new antiviral strategiesare very much needed. Semliki Forest virus (SFV) and Sindbis virus arehighly developed experimental paradigms for the alphavirus genus in partbecause of their low pathogenicity in humans.

The Class I viral fusion proteins: structure and inhibition. Membranefusion is a key step in the infection pathway of enveloped animalviruses. A number of virus membrane fusion proteins, exemplified by theinfluenza hemagglutinin (HA) and HIV gp41, share key features and aretherefore grouped together as class I fusion proteins (reviewed in Dutchet al., 2000; Eckert and Kim, 2001b; Weissenhorn et al., 1999). Membersof this class to date include orthomyxoviruses such as influenza (Skeheland Wiley, 2000), paramyxoviruses (Baker et al., 1999), retroviruses(Fass et al., 1996), filoviruses (Malashkevich et al., 1999),coronaviruses (Supekar et al., 2004) and human immunodeficiency virus-1(HIV-1) (Chan et al., 1997; Weissenhorn et al., 1997). Fusion of virusesin this class may be triggered by low pH as in the case of influenzavirus, by receptor interaction (Morrison, 2001), by receptor plus low pH(Hernandez et al., 1997; Mothes et al., 2000), or by receptor plusco-receptor interaction as in the case of HIV-1 (Binley and Moore, 1997;Feng et al., 1996). Class I fusion proteins are trimers that aregenerally proteolytically processed to produce a transmembranepolypeptide containing a hydrophobic sequence known as the fusionpeptide. Once fusion is triggered the hydrophobic fusion peptide istranslocated to the top of the molecule via formation of an extendedtrimeric coiled-coil α-helix (FIG. 1). The fusion peptide then insertsinto the target membrane and the protein refolds to form a “trimer ofhairpins” with a central α-helical coiled coil domain. Rearrangement tothis hairpin configuration repositions the fusion peptide andtransmembrane domains to the same end of a rod-like structure and drivesthe fusion reaction (Melikyan et al., 2000; Russell et al., 2001).Importantly, for a number of class I proteins, peptides containingsequences of the N or C-terminal interacting regions can bind to thefusion protein and inhibit fusion and infection by preventing refoldingto the final hairpin conformation (reviewed in Eckert and Kim, 2001a;Moore and Doms, 2003). This dominant-negative approach is exemplified bythe HIV peptide T20/Enfuvirtide, a licensed antiretroviral drug thatcorresponds to the C-terminal helix of gp41 (FIG. 1). While the firstclass I inhibitors were peptides, more recently small molecules thattarget critical sites of interaction in the trimer of hairpins have alsobeen shown to act as fusion inhibitors. One such small moleculeinhibitor of the paramyxovirus respiratory syncytial virus was recentlydemonstrated to bind to a hydrophobic pocket on the central coiled-coilof the fusion protein (Clanci et al., 2004). Thus, although they targetonly a subset of the contacts involved in forming the trimer ofhairpins, small molecules can act as potent inhibitors of hairpinformation and fusion. As small molecules can have higher bioavailabilitythan peptides, they provide an important approach to develop more widelyuseful fusion inhibitors, including the potential to target thoseviruses that fuse within endocytic compartments. Significantly, once theinitial proof of principle was established by the HIV T20 studies, theinhibition of virus fusion became an important focus of antiviralresearch for other class I viruses.

Class II viral fusion proteins: the alphavirus/flavivirus class. Thepre-fusion structures of the fusion protein ectodomains from thealphavirus Semliki Forest virus (SFV) (Lescar et al., 2001) and theflaviviruses dengue virus (Modis et al., 2003, 2005) and TBE virus (Reyet al., 1995) demonstrate that they are representative of a novel classof fusion proteins now termed “class II”. The SFV fusion protein (termedE1) and those of the flaviviruses (termed E) are highly similar inoverall fold and domain organization even though their primary sequencesare not conserved (FIG. 2). Both E1 and E are elongated moleculescontaining three domains, with the fusion peptide (orange) at the tip ofthe molecule in domain II (yellow), and the stem region andtransmembrane domain connecting to domain III (blue) at the oppositeend. The structures are composed predominantly of β-strand secondarystructure and contain no extended regions of α-helix or regionspredicted to form coiled-coils. Electron cryo-microscopy of virus andfitting of the structures shows that the fusion proteins lie tangential(almost parallel) to the virus membrane and organize in an icosahedralscaffold (Lescar et al., 2001; Pletnev et al., 2001; Zhang et al.,2002a).

The alpha- and flavivirus membrane fusion reactions are induced by lowpH within the endocytic pathway, which triggers the formation of ahighly stable homotrimer (HT) of the fusion protein. All of theavailable evidence indicates that, analogous to formation of the class Ihairpin, formation of the class II fusion protein HT is critical for thefusion reaction (Allison et al., 1995; Kielian et al., 1996; Wahlbergand Garoff, 1992). We developed methods to produce a proteolyticallytruncated ectodomain fragment of the SFV fusion protein E1 from purifiedvirus (Gibbons and Kielian, 2002; Kielian and Helenius, 1985). We alsodeveloped a method to convert the soluble ectodomain, termed E1*, to themembrane-inserted homotrimer form by treatment of the protein at acid pHin the presence of cholesterol-containing liposomes (Klimjack et al.,1994). This E1* homotrimer was then solubilized, purified, crystallizedand the homotrimer structure determined in collaboration with Dr. FélixRey of the CNRS (Gibbons et al., 2004a; Gibbons et al., 2004b).Strikingly, during trimerization domain III (blue) and the stem regionof E1* move about 37 Å towards the fusion peptide, and interact with thecentral core of the trimer (FIG. 3). Thus, although the E1* trimer doesnot contain an inner coiled-coil like the class I proteins, it forms amechanistically similar “hairpin” structure: a highly stable protein rodwith fusion peptide and transmembrane domains at the same end. Thestructures of the dengue virus and TBE homotrimers are remarkablysimilar to that of SFV (Bressanelli et al., 2004; Modis et al., 2004).Together the structural data demonstrate that the alphavirus andflavivirus membrane fusion proteins share common structural andfunctional features in both their pre-fusion and post-fusionconformations. Thus, the flaviviruses and alphaviruses form theinaugural members of the class II virus fusion proteins. We anticipatethat, similar to the class I proteins, other viral fusion proteins willultimately be assigned to class II as information about their structuresbecomes available. For example, modeling studies suggest that the HCV E2protein may also be a class II virus fusion protein (Yagnik et al.,2000). Bunyaviridae also apparently has class II virus fusion proteins(Garry and Garry, 2004).

Life cycle of the alphaviruses and flaviviruses. Both the alphavirusesand flaviviruses are small spherical enveloped viruses containingplus-strand RNA genomes packaged with a capsid protein (reviewed inLindenbach and Rice, 2001; Schlesinger and Schlesinger, 2001). The viralenvelope contains the transmembrane fusion protein (E1 or E) and asecond accessory or companion transmembrane protein (alphavirus E2 orflavivirus M). Both E2 and M are synthesized as larger precursorproteins (p62 and prM, respectively) that are cleaved by the cellularprotease furin late in the exocytic pathway. During biosynthesis, thefusion protein and companion protein associate within the endoplasmicreticulum (ER) to form non-covalent heterodimers, a process required forproper folding and transport of the fusion protein. Viruses containinguncleaved p62 or prM have greatly reduced infectivity and fusion due tothe interaction of the uncleaved companion subunit with the fusionprotein to prevent HT formation (Heinz et al., 1994; Salminen et al.,1992). In the mature alphavirus, the E2 protein maintains a stableheterodimer interaction with E1 and forms most of the projecting “spiky”domain of the alphavirus envelope (Zhang et al., 2002a). In contrast,the mature flavivirus particle is almost smooth as most of theectodomain of the prM protein is removed by furin (Zhang et al., 2003c).Following prM processing, the flavivirus E protein forms an E-Ehomodimer. Budding of the alphaviruses occurs at the plasma membrane andis dependent on the interaction of the E2 cytoplasmic tail with theviral nucleocapsid. Flaviviruses bud into the ER and budding can produceeither complete virions or smaller subviral (nucleocapsid-deficient)particles (reviewed in Garoff et al., 1998; Garoff et al., 2004; Strausset al., 1995).

The virus entry pathway has been most fully characterized for thealphaviruses but appears very similar for the flaviviruses (reviewed inHeinz and Allison, 2001; Kielian, 1995; Kielian et al., 2000). Virusbinds to receptors on the plasma membrane via the alphavirus E2 proteinor the domain III region of the flavivirus E protein. The virus isinternalized by the constitutive process of endocytosis. Virus-membranefusion is triggered by the mildly acidic pH of the endosome compartment,with a threshold pH of ˜6.2 for wt SFV. This low pH causes thedissociation of the alphavirus E2/E1 heterodimer or the flavivirus E/Ehomodimer. The monomeric E1 or E protein then inserts into the targetmembrane and refolds to form the homotrimer. Homotrimer formation,fusion, and infection are specifically blocked by various inhibitors ofendosome acidification, which act by raising the pH above the criticalfusion threshold (Glomb-Reinmund and Kielian, 1998; Kielian, 1995).Infection is also specifically blocked by expression ofdominant-negative versions of cellular proteins involved in theendocytic entry pathway (e.g., Sieczkarski and Whittaker, 2002;Sieczkarski and Whittaker, 2003).

The fusion of SFV, Sindbis, and TBE with liposomes is efficientlytriggered by low pH treatment in vitro, and is strongly promoted by thepresence of cholesterol in the target membrane (Corver et al., 2000;Kielian and Helenius, 1984; Smit et al., 1999; Stiasny et al., 2003).This sterol requirement is also observed in vivo usingcholesterol-depleted insect cells, and involves the sterol 3β-hydroxy 1group rather than the bulk physical effects of cholesterol (Lu et al.,1999; Phalen and Kielian, 1991; Vashishtha et al., 1998). The solublefusion protein ectodomains of SFV, dengue, and TBE form homotrimers thatare biochemically comparable to those formed by the full-lengthmolecules (Gibbons and Kielian, 2002; Klimjack et al., 1994; Stiasny etal., 2002), which made possible the structural studies discussed above.The membrane insertion and trimerization of the ectodomains requires thepresence of cholesterol in the target membrane (Klimjack et al., 1994),and is highly cooperative, producing rings of 5-6 homotrimers (Gibbonset al., 2003; Stiasny et al., 2004).

Other references relating to the present invention are Modis et al.,2003; Modis et al., 2004; Modis et al, 2005; Zhang et al., 2004;Vashishtha et al., 1998; Hung et al., 2004; Hilgard and Stockert, 2000;Jaiswal et al., 2004; Volk et al., 2004; Wu et al., 2003; Lakowicz,1999; Owicki, 2000, and Ahn et al., 2002.

Summary. It is now clear that there is a second class of virus membranefusion proteins exemplified by the alphaviruses and flaviviruses.Although markedly different in structure from the class I fusionproteins, the class II proteins also form a hairpin structure with thefusion peptide and transmembrane domains at the same end of a highlystable protein rod.

Due to the importance of viruses having class II fusion proteins, itwould be desirable to further characterize the fusion process in theseviruses and to identify ways to inhibit fusion. The present inventionaddresses that need.

SUMMARY OF THE INVENTION

The present invention is based in part on the inventors' discovery thataddition of a soluble Domain III of a virus having a class II virusfusion protein to a eukaryotic cell inhibits infection of the cell bythe virus and related viruses.

Accordingly, the present invention is directed to methods of inhibitingviral infection of a eukaryotic cell by a target virus having a class IIvirus fusion protein. The methods comprise combining the virus with anaqueous-soluble protein comprising a domain equivalent to a Domain IIIof an Alphavirus fusion protein, where the Domain III has SEQ ID NO:1.

In other embodiments, the invention is directed to methods of screeninga test compound for the ability to inhibit infection by a virus having aclass II viral fusion protein, the method comprising

(a) combining the test compound with

-   -   (i) an aqueous-soluble protein comprising a domain equivalent to        a Domain III of an Alphavirus fusion protein, wherein the Domain        III has SEQ ID NO:1, and    -   (ii) a core homotrimer of the class II viral fusion protein,        then

(b) determining whether the aqueous-soluble protein and the core trimerare bound together. In these methods, reduced binding of the proteinwith the core homotrimer in the presence of the test compound indicatesthat the test compound inhibits infection by the virus.

The present invention is also directed to other methods of screening atest compound for the ability to inhibit infection by a virus having aclass II viral fusion protein. These methods comprise

(a) combining the test compound with a class II viral fusion protein,

-   -   where the class II viral fusion protein comprises a Domain I, a        Domain II, and a Domain III, and where the Domain III portion        further comprises a fluorescent molecule that is quenched upon        fold-back of the Domain III region during trimerization, and

(b) determining whether the test compound reduces quenching of thefluorescent molecule upon conditions where quenching occurs in theabsence of the test compound. In these methods, reduced quenching of thefluorescent molecule indicates that the test compound inhibits infectionby the virus.

In further embodiments, the invention is directed to aqueous-solubleproteins comprising a portion of a class II viral fusion protein,wherein the portion of the class II viral fusion protein comprises adomain equivalent to a Domain III of an Alphavirus fusion protein,wherein the Domain III has SEQ ID NO:1, and a region equivalent to atleast a portion of a stem region of an Alphavirus fusion protein,wherein the stem region has SEQ ID NO:2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the inhibition of HIV fusion by T20 peptide. T20acts to inhibit the critical refolding reaction that drives HIV-1membrane fusion (schematic courtesy of Dr. R. Doms).

FIG. 2 is a ribbon model of the structure of the SFV E1 and TBE Eproteins, from Lescar et al., 2001.

FIG. 3 is diagrams of SFV E1 HT. On the left, a ribbon model of a lowpH-induced SFV E1* trimer structure showing model for membrane insertion(Gibbons et al., Nature 2004). On right is a schematic of the final.postfusion conformation of SFV E1 trimer showing fusion peptide (star),stem, and TM domains.

FIG. 4 shows a schematic of target protein strategies to follow theinteraction of fluorescent dIII (light grey ball) with core HT.Interaction of dIII is shown either (1) during trimerization or (2)post-trimerization.

FIG. 5 is a schematic model for inhibition of SFV fusion by addition ofsoluble domain III.

FIG. 6 is diagrams, photographs of autoradiographs, and a chartsummarizing domain III proteins discussed in the Example. Panel A showslinear diagrams of primary sequences of SFV E1, DV2 E and domain IIIconstructs, showing the boundaries of the domains, stem region, andtransmembrane anchor region (TM). The SFV E1 domain III proteins are:DIII (residues 291-383), DIIIS (291-412), His-DIII (His-tag plus291-383) and His-DIIIS (His-tag plus 291-412); the DV2 E domain IIIproteins are: DV2DIIIH1 (296-415) and His-DV2DIII (His-tag plus296-395). The His-tag adds 36 residues at the N-terminus while untaggedproteins contain an added methionine at the N-terminus. Panel B showsexperimental results where 2 μg of each purified domain III protein wastreated with or without 10 mM DTT, then alkylated and subjected toelectrophoresis on a 16.5% acrylamide gel using a Tris-tricine buffersystem. Marker proteins are shown on the left with their molecularmasses listed in kilodaltons. Panel C is a chart of experimental resultswhere the molecular mass of each domain III protein was measured by massspectrometry and compared with that calculated from the primary aminoacid sequence. The mass for DV2DIIIH1 was calculated without the addedN-terminal methionine since the measured mass indicated that thisresidue was not contained in the protein.

FIG. 7 is graphs of experimental results showing that SFV E1 domain IIIproteins inhibited SFV fusion with target cell membranes. In theexperiment summarized in Panel A, SFV was added to BHK cells (MOI˜0.002) for 90 min on ice (binding). The cells were then incubated at pH7.4 (indicated as “N”) or at pH 5.5 at 37° C. for 1 min (fusion), andcultured at 28° C. overnight in medium containing 20 mM NH₄Cl (culture).The presence or absence of 4 μM His-DIII in each step is indicated by+/−. Infected cells were quantitated by immunofluorescence. Results areshown as percent of control infection in the absence of any addedHis-DIII. Panel B shows the concentration dependence of domain IIIinhibition as determined using the assay in Panel 7A and adding theindicated concentrations of domain III proteins only during the 1 minlow pH treatment. Data are a representative example from 2 independentexperiments.

FIG. 8 is graphs of experimental results showing the inhibitory effectof domain III proteins on class II virus fusion, which shows broadspectrum and specificity inside the virus genus.

FIG. 9 is a graph of experimental results where SFV E1 domain IIIproteins inhibited SFV and SIN (Sindbis virus, an alphavirus) infectionin the endocytic pathway. SFV, SIN, VSV and DV2 were diluted in mediumof pH 7.2 containing the indicated concentrations of domain III. Viruseswere incubated with BHK cells for 1 h at 20° C. to allow endocyticuptake. Infection was blocked by addition of medium containing 20 mMNH₄Cl, the cells were incubated overnight and infected cells quantitatedby immunofluorescence. Data are shown as percent of control infection inthe absence of domain III, and are the average of 3 independentexperiments.

FIG. 10 is a fluorescence scan and a graph showing experimental resultswhere SFV E1 domain III proteins inhibited the lipid mixing step offusion. Panel A is a fluorescence scan of pyrene-labeled SFV fused withBHK cells. Pyrene-labeled SFV was pre-bound to BHK cells and incubatedat 37° C. for 1 min in pH 7.4 medium without domain III protein (curvea), in pH 5.5 medium without domain III protein (curve b), or in pH 5.5medium with 1 μM (curve c), 5 μM (curve d) or 8 μM (curve e) His-DIIIS.Background fluorescence from cells alone was subtracted and thefluorescence emission was normalized for each sample by setting themonomer peak at 397 nm to 5 (arbitrary units). Data shown are arepresentative example from 3 independent experiments. Panel B is agraph showing a comparison of inhibition of lipid mixing by domain IIIproteins. The fusion between pyrene-labeled SFV and BHK cells wasassayed as in Panel 10A in the presence of the indicated concentrationsof domain III proteins. The difference between the excimer/monomer(Ex/M) ratio at pH 7.4 and after treatment at pH 5.5 without domain IIIwas defined as 100% (control). The difference between the pH 7.4 sampleand each experimental sample was determined and expressed as a percentof this control difference. Data shown are the average from 3independent experiments.

FIG. 11 is photographs of autoradiographs and a graph of experimentalresults showing that SFV domain III proteins bind to trimeric E1 duringthe fusion reaction. In the experiment summarized in Panel A,³⁵S-labeled SFV was bound to BHK cells on ice and treated at pH 7.4 or5.5 at 37° C. for 1 min in the presence of the indicated domain IIIproteins. Cells were then lysed and immunoprecipitated with a rabbitpolyclonal antibody against the SFV E1 and E2 protein (Rab), a mAbagainst the low pH conformation of E1 (E1a-1), a mAb against the His-tag(HIS-1), rabbit pre-immune serum (Pre), or an isotype-matched irrelevantmAb (12G5). Samples were analyzed by SDS-PAGE and fluorography. Panel Bis a graph showing quantitation of samples prepared as in 11A using theindicated concentrations of His-DIII or His-DIIIS. “N” indicates 1 mintreatment at pH 7.4 with 2 μM His-DIIIS. The total E1 in each sample wasdefined as the amount of E1 immunoprecipitated by RAb. Data are arepresentative example of 2 independent experiments. Panel C isautoradiographs showing that exogenous domain III proteins decreased theamount of SDS-resistant E1 homotrimer. Samples were prepared as withPanel 11B. An aliquot of the cell lysate was treated with SDS-samplebuffer at 30° C. and analyzed by SDS-PAGE and fluorography. The positionof the SDS-resistant homotrimer is indicated. Panel D is anautoradiograph showing that domain III selectively interacts with atrimeric form of E1. Fusion reactions were induced at pH 7.4 or pH 5.5in the presence of 10 μM His-DIII as in Panel 11A. Samples wereimmunoprecipitated with the indicated antibodies and then digested withtrypsin as indicated. The amount of trypsin-resistant E1 was quantitatedand expressed as a percent of the non-trypsinized E1 for each sample.Data shown are a representative example of 2 independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the inventors' discovery thataddition of a soluble Domain III of a virus having a class II virusfusion protein to a eukaryotic cell inhibits infection of the cell bythe virus and related viruses. See Liao and Kielian, 2006 and Examplebelow. Without being bound by any particular mechanism, it is believedthat the soluble Domain III binds to the class II virus fusion proteinpreventing foldback of the fusion protein Domain III duringtrimerization. The inventors have used this discovery to develop novelmethods for inhibiting viral infection and for determining whether atest compound has the ability to inhibit viral infection. Novelcompositions useful for these methods are also provided.

Accordingly, the present invention is directed to methods of inhibitingviral infection of a eukaryotic cell by a target virus having a class IIvirus fusion protein. The methods comprise combining the virus with anaqueous-soluble protein comprising a domain equivalent to a Domain IIIof an Alphavirus fusion protein, where the Domain III has SEQ ID NO:1.

As used herein, a “domain” is a discrete portion of a protein having itsown function and characteristic three-dimensional structure. A DomainIII (“dIII”, “domain III”) is a recognized domain of a class II fusionprotein with a structure and function equivalent to the domain IIIregion of the Semliki Forest virus (SFV) class II fusion protein (E),where the domain III has the sequence of SEQ ID NO:1. A Domain IIIinteracts with the central core of the trimer upon trimerization whenpart of a functional class II fusion protein. It comprises a portion ofnear the C-terminal domain of the SFV fusion protein. A Domain III isgenerally expected to have a ribbon structure substantially as depictedin FIG. 2, as shown for SFV and tick-borne encephalitis virus (TBE). Theamino acid sequence need not have any homology with SEQ ID NO:1, butmust only be part of a naturally-occurring class II fusion protein, or aderivative thereof, where the domain III interacts with the central coreof the trimer upon trimerization. Thus, mutants of a naturally occurringdomain III are envisioned as within the scope of a domain III, providedthe mutant domain, if part of a class II fusion protein, is stillcapable of interacting with the central core of the trimer upontrimerization

These methods are useful for inhibiting infection of any eukaryotic cellsusceptible to a virus having a class II fusion protein, including plantcells and mammalian cells, e.g., human cells. Preferably, the cell ispart of a living multicellular eukaryote, more preferably a mammal, andmost preferably a human.

As used herein, “aqueous-soluble” means soluble in an aqueous solutionand/or suspension, e.g. water, saline, buffered saline, and/or aphysiological fluid such as blood, bile, or lymph.

The aqueous-soluble protein in these embodiments can comprisepeptidomimetics as substitutes for one or more than one amino acidmoiety. As used herein, an amino acid mimetic or peptidomimetic is acompound that is capable of mimicking a natural parent amino acid in aprotein, in that the substitution of an amino acid with thepeptidomimetic does not affect the activity of the protein. Proteinscomprising peptidomimetics are generally poor substrates of proteasesand are likely to be active in vivo for a longer period of time ascompared to the natural proteins. In addition, they could be lessantigenic and show an overall higher bioavailability. The skilledartisan would understand that design and synthesis of aqueous-solubleproteins comprising peptidomimetics would not require undueexperimentation. See, e.g., Ripka et al., 1998; Kieber-Emmons et al.,1997; Sanderson, 1999.

In some preferred embodiments, the aqueous-soluble protein furthercomprises a region equivalent to at least a portion of a stem region ofan Alphavirus fusion protein, wherein the stem region has SEQ ID NO:2.As shown in the Example, retaining on the aqueous-soluble protein atleast a portion of the stem region of a class II fusion protein canimprove the ability of the aqueous-soluble protein to inhibit infection.As used herein, the stem region of a class II fusion protein is theregion equivalent to the stem region of SFV, having the sequence of SEQID NO:2. The stem region of SFV is 29 amino acids immediately toward thecarboxy end of the fusion protein from the Domain III. In theseembodiments, an equivalent stem region need not have homology to SEQ IDNO:2, but will be structurally similar. The skilled artisan couldidentify a stem region of any class II fusion protein without undueexperimentation.

The aqueous-soluble protein preferably also further comprises a regionequivalent to a DI/DIII linker region from an Alphavirus class II fusionprotein, where the Alphavirus linker region has SEQ ID NO:11. The linkerregion defined herein as SEQ ID NO:11 is somewhat smaller than thelinker region as identified in Roussel et al., 2006. The inclusion ofthis linker region also improves binding of the aqueous-soluble proteinto the homotrimer. The skilled artisan could identify a linker region ofany class II fusion protein without undue experimentation.

In additional embodiments, the aqueous-soluble protein further comprisesan oligohistidine moiety (e.g., a His6). Such moieties are known to aidin purification of proteins that are engineered to have them, by virtueof their ability to bind to certain chromatography media. An example ofan engineered His6 moiety is the first thirty-six residues of SEQ IDNO:7.

In some preferred embodiments of these methods, the aqueous-solubleprotein comprises SEQ ID NO:1. The aqueous-soluble protein can bepresent in any concentration that effects inhibition of virus infection.As shown in the Example, that concentration can be as low as 50 nM, 100nM, 250 nM, 500 nM, 1000 nM, 5000 mM, or greater.

The above-described aqueous-soluble proteins can be formulated withoutundue experimentation into a pharmaceutical composition foradministration to a mammal, including humans, as appropriate for theparticular application. Additionally, proper dosages of the compositionscan be determined without undue experimentation using standarddose-response protocols.

Accordingly, the compositions designed for oral, lingual, sublingual,buccal and intrabuccal administration can be made without undueexperimentation by means well known in the art, for example with aninert diluent or with an edible carrier. The compositions may beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral therapeutic administration, the pharmaceutical compositions ofthe present invention may be incorporated with excipients and used inthe form of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, andflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth or gelatin. Examples of excipients includestarch or lactose. Some examples of disintegrating agents includealginic acid, corn starch and the like. Examples of lubricants includemagnesium stearate or potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring and the like. Materialsused in preparing these various compositions should be pharmaceuticallypure and nontoxic in the amounts used.

The compositions of the present invention can easily be administeredparenterally such as for example, by intravenous, intramuscular,intrathecal or subcutaneous injection. Parenteral administration can beaccomplished by incorporating the compositions of the present inventioninto a solution or suspension. Such solutions or suspensions may alsoinclude sterile diluents such as water for injection, saline solution,fixed oils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents. Parenteral formulations may also includeantibacterial agents such as for example, benzyl alcohol or methylparabens, antioxidants such as for example, ascorbic acid or sodiumbisulfite and chelating agents such as EDTA. Buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose may also be added. The parenteralpreparation can be enclosed in ampules, disposable syringes or multipledose vials made of glass or plastic.

Rectal administration includes administering the pharmaceuticalcompositions into the rectum or large intestine. This can beaccomplished using suppositories or enemas. Suppository formulations caneasily be made by methods known in the art. For example, suppositoryformulations can be prepared by heating glycerin to about 120° C.,dissolving the composition in the glycerin, mixing the heated glycerinafter which purified water may be added, and pouring the hot mixtureinto a suppository mold.

Transdermal administration includes percutaneous absorption of thecomposition through the skin. Transdermal formulations include patches(such as the well-known nicotine patch), ointments, creams, gels, salvesand the like.

The present invention includes nasally administering to the mammal atherapeutically effective amount of the composition. As used herein,nasally administering or nasal administration includes administering thecomposition to the mucous membranes of the nasal passage or nasal cavityof the patient. As used herein, pharmaceutical compositions for nasaladministration of a composition include therapeutically effectiveamounts of the composition prepared by well-known methods to beadministered, for example, as a nasal spray, nasal drop, suspension,gel, ointment, cream or powder. Administration of the composition mayalso take place using a nasal tampon or nasal sponge.

These methods are expected to be useful for inhibiting infection for anyvirus having a class II fusion protein, including any such virus nowknown or later discovered. Class II fusion proteins are now known in theAlphavirus (e.g., Semliki Forest virus) (family Togaviridae), Flavivirus(e.g., Dengue virus) (family Flaviviridae), Hepacivirus (e.g., hepatitisC virus) (family Flaviviridae), and Phlebovirus (e.g., Sandfly fevervirus) (family Bunyaviridae) (Garry and Garry, 2004). All members of theTogaviridae, Flaviviridae, and Bunyaviridae would therefore be expectedto have type II fusion proteins. Additionally, while Togaviridae andFlaviviridae are positive strand RNA viruses, Bunyaviridae are negativestrand RNA viruses. Further, Rhabdoviridae may also have type II fusionproteins. This indicates that viruses having class II fusion proteinsare widespread.

Thus, in some embodiments of these methods, the target virus is anAlphavirus, for example Semliki Forest virus, eastern equineencephalitis virus, western equine encephalitis virus, or Venezuelanequine encephalitis virus. In other embodiments, the target virus is amember of the Flaviviridae, such as dengue virus, hepatitis C virus,Japanese encephalitis virus, tick-borne encephalitis virus, yellow fevervirus, West Nile virus, bovine viral diarrhea virus, or swine fevervirus. Additionally, the target virus can be a member of theBunyaviridae, for example Crimean-Congo hemorrhagic fever virus ortomato spotted wilt virus. In some preferred embodiments, the targetvirus is Semliki Forest virus or dengue virus.

In preferred embodiments, the aqueous-soluble protein is a portion ofthe class II virus fusion protein of a virus in the same viral genus asthe target virus. In more preferred embodiments, the aqueous-solubleprotein is a portion of the class II virus fusion protein of the targetvirus.

These methods can be utilized in vitro (e.g., in cells in culture), orpreferably, where the target virus is in a living eukaryote. In somepreferred embodiments, the living eukaryote is a mammal.

The discovery of the effect the interaction of Domain III with the restof the class III fusion protein, and the role of that interaction inviral infection, has led the inventors to develop assays for screeningtest compounds for the ability to inhibit viral infection.

Thus, the invention is also directed to methods of screening a testcompound for the ability to inhibit infection by a virus having a classII viral fusion protein. The methods comprise

(a) combining the test compound with

-   -   (i) an aqueous-soluble protein comprising a domain equivalent to        a Domain III of an Alphavirus fusion protein, where the Domain        III has SEQ ID NO:1, and    -   (ii) a core homotrimer of the class II viral fusion protein,        then

(b) determining whether the aqueous-soluble protein and the core trimerare bound together. In these embodiments, reduced binding of the proteinwith the core homotrimer in the presence of the test compound indicatesthat the test compound inhibits infection by the virus. Thus, theseassays determine whether the test compound inhibits the interaction ofthe Domain III with the core homotrimer.

In some preferred embodiments, the aqueous-soluble protein furthercomprises a region equivalent to at least a portion of a stem region ofan Alphavirus fusion protein. In these embodiments, the stem region ofthe Alphavirus fusion protein has SEQ ID NO:2. In other preferredembodiments, the aqueous-soluble protein further comprises a regionequivalent to a DI/DIII linker region from an Alphavirus class II fusionprotein, In these embodiments, the Alphavirus linker region has SEQ IDNO:11.

In some cases, the presence of an oligohistidine moiety also strengthensthe interaction between the aqueous-soluble protein and the homotrimer.See Example. Thus, in some embodiments of these methods, theaqueous-soluble protein further comprises an oligohistidine moiety.

In other preferred embodiments of these methods, the aqueous-solubleprotein comprises SEQ ID NO:1.

In preferred embodiments, the aqueous-soluble protein is labeled with adetectable label, generally to provide a detectable means to determinewhether the aqueous-soluble protein and the core trimer are boundtogether. In these embodiments, the detectable label is a fluorescentmolecule, a radioactive atom, an enzyme, or an antigen not naturallyoccurring in the aqueous-soluble protein. Preferably, the detectablelabel is a fluorescent molecule, where the determination step canutilize a method such as fluorescence polarization to determine binding.Such fluorescence polarization methods are known in the art. Withfluorescent labels, the determination step can alternatively utilizefluorescence resonance energy transfer. FIG. 4 shows two strategies tofollow the interaction of fluorescent Domain III with the corehomotrimer.

In these embodiments, the core homotrimer need not be a complete or awild-type form. It can include any mutations that do not substantiallyaffect the ability of the Domain III to interact with it. It can alsoinclude peptidomimetics, as described above. Additionally, the corehomotrimer can comprise a deletion or deletions from the wild-type form,provided that the mutant homotrimer is still capable of interacting withthe Domain III. The core homotrimer can also be within an intact wildtype or mutant virus. In some preferred embodiments, the core homotrimerdoes not comprise a domain equivalent to a Domain III of an Alphavirusfusion protein.

The present methods can be adapted to high throughput formats as areknown in the art, e.g., using robotics. Additionally or alternatively,the methods can utilize immobilization of either the core homotrimer orthe aqueous-soluble protein on a solid matrix, for example on amembrane, a bead or in a well such as a polystyrene plate. Such methodscould be developed without undue experimentation.

Test compounds can also be screened for the ability to inhibit infectionby a virus having a class II fusion protein by utilizing a corehomotrimer with a Domain III portion that comprises a fluorescentmolecule in such a position that the fluorescence of the fluorescentmolecule is quenched upon fold-back of the Domain III upon trimerizationof the fusion protein. A compound that prevents such quenching inhibitstrimerization and infection. Alternatively, the fluorescence of thedomain III portion may be protected from an added aqueous quencher upontrimerization. A compound that prevents such protection inhibits domainIII foldback and infection.

Thus, in additional embodiments, the present invention is directed tomethods of screening a test compound for the ability to inhibitinfection by a virus having a class II viral fusion protein. The methodscomprise

(a) combining the test compound with a class II viral fusion protein,

-   -   where the class II viral fusion protein comprises a Domain I, a        Domain II, and a Domain III, and wherein the Domain III further        comprises a fluorescent molecule that is quenched upon fold-back        of the Domain III, and

(b) determining whether the test compound reduces quenching of thefluorescent molecule upon conditions where quenching occurs in theabsence of the test compound. In these embodiments, reduced quenching ofthe fluorescent molecule indicates that the test compound inhibitsinfection by the virus.

Examples of residues of class II fusion proteins that could befluorescently labeled, where the fluorescence would be quenched upontrimerization, are residues equivalent to Asp311, Phe312, Thr 338,Gln340, or His 331 of a Semliki Forest Virus class II fusion protein orGln316 or His317 of a Dengue virus 2 class II fusion protein. Other suchresidues can be determined without undue experimentation by identifyingsuitable residues at the Domain III-core trimer interface.

The present invention is also directed to aqueous-soluble proteinscomprising a portion of a class II viral fusion protein, where theportion of the class II viral fusion protein comprises

-   -   (a) a domain equivalent to a Domain III of an Alphavirus fusion        protein, wherein the Domain III has SEQ ID NO:1, and    -   (b) a region equivalent to at least a portion of a stem region        of an Alphavirus fusion protein, wherein the stem region has SEQ        ID NO:2. Based on experimental results provided in the Example,        such novel proteins are useful in that they prevent infection of        eukaryotic cells by viruses with class II fusion proteins, and        can be utilized in the screening assays described above.

The aqueous-soluble protein of these embodiments also preferablycomprises a region equivalent to a DI/DIII linker region from anAlphavirus class II fusion protein. Here, the Alphavirus linker regionhas SEQ ID NO:11

In some preferred embodiments, these aqueous-soluble proteins compriseSEQ ID NO:1 and SEQ ID NO:2. Alternatively, the aqueous-soluble proteincan comprise mutations, deletions, peptidomimetics, etc.

The class II viral fusion protein of these embodiments can be derivedfrom any virus having such a protein. In preferred embodiments, theclass II viral fusion protein is from a member of the Togaviridae,Flaviviridae, or Bunyaviridae. In this regard, the class II viral fusionprotein can be from, e.g., an Alphavirus or a Flaviviridae.

Furthermore, the aqueous-soluble protein of these embodiments canfurther comprise a detectable label and/or a binding moiety, for examplea fluorescent molecule, a radioactive atom, an enzyme, an antigen notnaturally occurring in the aqueous-soluble protein, or an oligohistidinemoiety. In some preferred embodiments, the detectable label is afluorescent molecule; in other preferred embodiments the aqueous-solubleprotein comprises an oligohistidine moiety as discussed above.

Preferred embodiments of the invention are described in the followingexample. Other embodiments within the scope of the claims herein will beapparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims that follow the example.

Example Domain III from Class III Fusion Proteins Functions as aDominant-Negative Inhibitor of Virus Membrane Fusion

Rationale and domain III expression. The structure of the class IIhomotrimer suggests several features that might be targets forinhibition of the fusion reaction. The pH 7 form of the dengue virus Eprotein crystallized with a molecule of detergent bound in a hydrophobicpocket near the “hinge” region between domains 1 and II (Modis et al.,2003). As the hinge changes its angle during the transition to thetrimer form, inhibition of this flexibility may block fusion andinfection. Thus, molecules that dock into the hydrophobic pocket arepotential antivirals (Modis et al., 2003). The structure of the fusionprotein homotrimer reveals that the “stem” region of the proteininteracts (or would be predicted to interact in the case of the dengueHT and TBE HT, which do not contain the stem) with an HT core regioncomposed of domains I and II (see stem in FIG. 3 schematic). Therefore,the stem peptide and its HT interaction site are potential targets,analogous to the T20 peptide of HIV and its interaction site on thecentral coiled-coil (Bressanelli et al., 2004; Modis et al., 2004;Roussel et al., 2006; Kielian, 2006; Kielian and Rey, 2006). We havetested several stem peptides and antibodies to the stem region for theirability to inhibit Semliki Forest virus (SFV) HT formation and fusion.None of these reagents showed any inhibitory activity. Given thatidentification of peptide inhibitors of class I fusion reactionsfrequently requires screening of many candidate peptides, it iscertainly possible that class II stem peptides will eventually work asantiviral reagents. However, we felt that it was important to considerthe inhibitor question more broadly. The structure of the low pH-inducedtrimer revealed a striking movement of domain III towards the trimertip, resulting in the interaction of domain III with the HT core(composed of domains I and II in trimeric form). This reorientation ofdomain III appears to be a critical feature of the formation of theclass II hairpin. We therefore decided to test soluble forms of domainIII as potential inhibitors (FIG. 5).

Several previous studies had demonstrated that flavivirus domain IIIcould be produced in bacteria as fusion proteins (Volk et al., 2004),epitope-tagged proteins (Hung et al., 2004; Wu et al., 2003), or byrefolding of the expressed molecule (Jaiswal et al., 2004). Thestructures of recombinant domain III from West Nile virus (Volk et al.,2004) and Japanese encephalitis virus (Wu et al., 2003) were determinedby NMR and shown to be essentially identical to the structure of dIII inTBE E protein purified from virus (Rey et al., 1995). We took thisinformation into account in our expression of the analogous alphavirusprotein. SFV domain III is contiguous in the linear sequence of E1, andforms an Ig-like β-barrel structure with three disulfide bonds (FIG. 2).

We prepared four domain III constructs for SFV, containing domain IIIwith or without the stem region (DIIIS, DIII, respectively) and with orwithout an N-terminal His-tag (His-DIIIS, His-DIII) (FIG. 6A). We alsoprepared 2 constructs of domain III from the dengue 2 serotype:DV2DIIIH1 (DIII plus the helix 1 region of the stem) and His-DV2DIII(DIII with an N-terminal His tag). The proteins were expressed in E.coli, refolded using a fast dilution method successfully used to refolda number of proteins containing Ig-like domains (Zhang et al., 2002b),and purified by FPLC. All of the purified proteins migrate as a singleband of the predicted size in SDS-PAGE and show a mobility shift uponreduction indicating the presence of disulfide bond(s) (FIG. 6B). FPLCanalysis of the purified SFV proteins showed that they elute as singlepeaks at either neutral pH or pH 5.5 (data not shown). Analysis by massspectroscopy confirmed the predicted protein sizes and suggested thatthe SFV domain III constructs contain three disulfide bonds since theirdetermined masses are approximately 6 units less than those predicted ifall 6 cysteines are reduced) (FIG. 6C). Similarly, the dengue domain IIIconstructs appear to contain the single predicted disulfide. Thedisperse location of the cysteines in SFV domain III suggests that theycannot form aberrant disulfides without radically changing the proteinfold (Lescar et al., 2001). Thus the presence of all three disulfidebonds, the proteins' high solubility (>10 mg/ml), and the biologicalactivity described below strongly suggest that all of the SFV constructshave generated correctly folded domain III, similar to the preparationspreviously characterized for flavivirus domain III.

Inhibition of fusion and infection by SFV domain III preparations. Wethen screened the domain III preparations for inhibitory activity invirus fusion and infection assays. A simple, highly quantitative fusionassay extensively used by our lab is the fusion-infection assay (FIA)(Vashishtha et al., 1998; Zhang et al., 2003b). In this assay, serialdilutions of virus are added to cells on ice and allowed to bind in thecold. The unbound virus is washed away, and the cells with bound virusare treated briefly (1 min at 37° C.) at low pH to trigger the fusion ofthe virus with the plasma membrane of the cell. This fusion results invirus infection. The cells are then cultured overnight in the presenceof 20 mM NH₄Cl to prevent secondary infection, and the cells infecteddue to the low pH pulse are quantitated by immunofluorescence. Underthese conditions, we could test the effects of domain III preparationsduring the binding step, the fusion step, and the post-fusion culturestep.

As shown in FIG. 7A, 4 μM His-DIII almost completely inhibited SFVinfection of BHK cells, but only when present during the low pH-inducedfusion step. Similar results were obtained for His-DIIIS (data notshown). In agreement with studies showing that alphavirus-receptorinteraction is mediated by the E2 protein (Strauss and Strauss, 1994),His-DIII did not inhibit virus-cell binding as assayed by the FIA (FIG.7A) or by quantitation of cell-associated radiolabeled SFV (data notshown, and FIG. 11C below). Pre-incubation of the virus with His-DIII at37° C. at neutral pH had no effect on subsequent FIA (data not shown).Inhibition by domain III was comparable when virus was prebound to thecells at pH 6.5, 6.8, 7.4, or 8.0, or when the low pH pulse was at pH5.5 or 6.0 (data not shown). Comparison of the 4 SFV domain IIIconstructs showed that the strongest inhibition was obtained withHis-DIIIS (IC50 ˜0.1 μM), followed by His-DIII (IC50 ˜0.5 μM), DIIIS(IC50 ˜6 μM) and DIII which gave ˜60% inhibition at a concentration of˜80 μM (FIG. 7B). Thus, the presence of both the stem region and theN-terminal His-tag resulted in increased effectiveness. Whileenhancement by the stem region is suggested from the structure of thelow pH-induced homotrimer, the reason for the increase in inhibitionobserved with the His-tagged protein is not known, and presumablyreflects a stabilization of the domain III-E1 interaction, as discussedbelow.

The specificity of inhibition was addressed by comparing the effect ofSFV domain III on fusion of the alphavirus Sindbis (SIN) and theflavivirus dengue 2 (DV2). The sequence of domain III is ˜50% identicalbetween SFV and SIN, while the DV2 E protein shows no detectablesequence conservation with the alphavirus fusion proteins overall or inthe domain III region. SFV, SIN, and DV2 all showed efficient fusionupon treatment at pH 5.5, and little fusion at pH 7.4 (FIG. 8A).Inclusion of SFV His-DIII or His-DIIIS during the low pH pulse inhibitedSIN fusion with comparable (or even slightly higher) efficiency as SFVfusion. Neither SFV domain III preparation caused any inhibition of DV2fusion.

To address the general applicability of domain III inhibition to classII fusion, we used the His-DV2DIII and DV2DIIIH1 protein preparationsand tested their ability to inhibit fusion by the DV2 and DV1 serotypesof dengue virus. These two serotypes show ˜60% sequence identity indomain III. Unlike alphaviruses, flavivirus-receptor binding is directlymediated by the membrane fusion protein, and maps primarily to domainIII. Prior studies of flavivirus domain III showed that it could blockvirus-cell binding when added prior to or concurrently with virus. Wetherefore prebound DV1 and DV2 to cells in the cold and added domain IIIonly during the one minute pH pulse used to trigger fusion. As shown inFIG. 8B, the DV2DIIIH1 protein strongly inhibited both DV1 and DV2fusion (˜70% inhibition at a concentration of 50 μM), but showed noactivity against SFV. Interestingly, His-DV2DIII did not inhibit fusionsuggesting a possible role for the helix 1 stem region. Treatment at 37°C. for 1 min with either DV domain III preparation did not release boundvirus from the cell membrane, implying that domain III inhibition wasnot due to effects on virus-receptor interaction (data not shown).Together these results strongly suggest that inhibition by DV2DIIIH1occurred by effects on membrane fusion rather than by effects on virusbinding. Thus, domain III can act as a specific inhibitor of the classII membrane fusion reaction. The observed cross-inhibition within thealphaviruses and flaviviruses suggests that key domain III amino acidcontacts may be conserved within each virus genus.

Since alphavirus-receptor binding is not mediated by the E1 protein, weused this system to test the ability of domain III proteins to inhibitvirus fusion from within the endosome, the physiological route of virusinfection. We infected BHK cells with SFV, SIN, vesicular stomatitisvirus (VSV), or DV2 in the presence or absence of 20 μM His-DIIIS or 40μM DIIIS. VSV, an unrelated rhabdovirus, and DV2 are important controlssince these viruses also infect cells by endocytosis and lowpH-triggered fusion (Matlin et al., 1982). After a 1 hour endocyticuptake period, 20 mM NH₄Cl was added to prevent further infection, andthe primary infected cells were quantitated by overnight culture andimmunofluorescence as described above. As shown in FIG. 9, infection byboth alphaviruses was significantly inhibited by the inclusion of eitherthe His-tagged or untagged forms of domain III plus stem. In contrast,VSV and DV2 infection were not inhibited, and DV2 infection was actuallyenhanced somewhat by DIIIS. Inhibition of alphavirus endocytic infectionby His-DIIIS required a higher concentration than in the FIA, and alsoshowed less efficacy versus untagged DIIIS than in the FIA. This couldreflect relatively inefficient endocytic uptake of His-DIIIS by thecells or differential routing of virus and domain III within theendocytic pathway. While targeting of domain III to the endosomal siteof virus fusion is probably not optimized, it is already clear thatdomain III can block fusion and infection under physiological virusentry conditions.

Exogenous domain III blocks the initial mixing of the virus and cellmembranes. Class II virus fusion initiates through the interaction ofthe fusion loop with the target membrane, and progresses through aninitial lipid mixing stage termed hemifusion, in which the outerleaflets of the virus and target membranes mix (Zaitseva et al., 2005).This stage is followed by the opening of a fusion pore, which thenwidens to give complete fusion and content mixing, the end stage offusion monitored by the FIA. To test for the effects of domain III oninitial lipid mixing and hemifusion, we followed the loss of the pyreneexcimer peak upon fusion of pyrene-labeled SFV with unlabeled targetcells (Chatterjee et al, 2002). Pyrene-labeled SFV was bound to cells inthe cold and pulsed at low pH in the presence or absence of domain III.We then determined the fluorescence emission spectrum of each virus-cellmixture and compared the ratio of the excimer and monomer peaks(Chatterjee et al, 2002). Untreated virus (data not shown) or virustreated at pH 7.4 showed a strong excimer peak, with an excimer tomonomer ratio of ˜0.28 (FIG. 10A, curve a). Virus treated at pH 5.5showed efficient fusion with the cell plasma membrane, as reflected inthe decrease of the excimer peak and an excimer/monomer ratio of ˜0.10(curve b). The presence of His-DIIIS caused a concentration-dependentinhibition of the lipid mixing step (curves c-e). No effect was observedwhen His-DIIIS was added to the sample after low pH treatment (data notshown). As observed in the FIA, the His-DIIIS form of domain III showedthe highest activity, with >90% inhibition of fusion at 8 μM (FIG. 10B).Both His-DIII and DIIIS showed significant inhibition at 20 μM, whilethe dengue virus protein showed no inhibition at 20 μM. A higherconcentration of His-DIIIS was required to completely inhibit pyrenevirus fusion compared to the FIA, reflecting either an intrinsicdifference in the inhibitor sensitivity of lipid mixing vs. contentmixing, or the higher concentration of virus used in the pyrene vs. FIAexperiments.

Interaction of domain III with E1 during fusion. If domain III isinhibiting virus fusion by preventing the fold-back of the full-lengthviral E1, it may interact stably with the E1 protein during inhibition.To assay for such interaction, we used radiolabeled SFV and His-DIII orHis-DIIIS in the FIA. Following the low pH-treatment step, the cellswere lysed in the non-ionic detergent octyl-glucoside, which we haveshown fully solubilizes membrane-inserted E1, disrupts inter-trimerinteractions, and maintains trimer structure (Gibbons, 2004a). Aliquotsof the samples were immunoprecipitated using either a polyclonalantibody to quantitate the total E1 and E2 proteins, monoclonal antibodyE1a-1, which specifically recognizes the acid-conformation of E1 (Ahn etal., 1999), an antibody that recognizes the His-epitope on domain III,or two control antibodies. SDS-PAGE demonstrated equivalent amounts ofradiolabeled virus proteins present in cells treated at neutral or lowpH with or without domain III, confirming that domain III does notrelease bound virus from the cell (FIG. 11A). Upon acid treatment the E1subunit was efficiently recognized by the acid-specific monoclonalantibody. Inclusion of either His-DIII and His-DIIIS during low pHtreatment resulted in co-immunoprecipitation of the E1 protein by theantibody to the His-tag. Similar to the inhibition of fusion activity,the interaction with E1 occurred only when domain III was present duringthe low pH treatment step, and not at neutral pH. The His-DIIIS proteinpreparation showed more efficient co-immunoprecipitation than His-DIII,in keeping with the more efficient inhibition of fusion by thestem-containing form of E1.

Quantitative analysis showed that the amount of E1 retrieved by theanti-His tag antibody increased when increasing amounts of domain IIIwere present during the low pH step (FIG. 11B). His-DIII interactionretrieved about 18% of the total E1 at a concentration of 20 μM.Retrieval by His-DIIIS was maximal at 2 μM and ˜50% of the total E1,similar to the amount of E1 that converted to reactivity with theacid-specific antibody. Interestingly, concentrations of His-DIIIS above2 μM led to a gradual decrease in the retrieval of E1 by both theanti-His antibody and the acid-conformation specific antibody. This isthe result that would be predicted if the presence of a highconcentration of His-DIIIS is directly affecting the HT. We evaluatedthis possibility by quantitating the E1 HT band in SDS-PAGE, takingadvantage of its relative resistance to dissociation by SDS samplebuffer at 30° C. (FIG. 11C). Increasing amounts of His-DIIIS lead to theloss of the HT band, with only 10% of the control HT observed in thepresence of 20 μM His-DIIIS. Thus, the presence of His-DIIIS interfereswith the formation or stability of the E1 HT. Interestingly, addition ofHis-DIIIS produced bands migrating above and below the position of theHT, suggesting the presence of alternative E1 complexes (FIG. 11C). Adecrease in the amount of E1HT was also observed in the presence ofincreasing amounts of His-DIII (60% of control HT at 20 μM His-DIII).

The target for domain III binding on E1 could be either the E1 monomerprior to trimerization, or a trimeric form of E1. A general property oftrimeric E1 is its relative resistance to trypsin digestion, which ismaintained even for E1 mutants that do not produce an SDS-resistant HT(Chatterjee et al., 2002). We treated cell-bound radiolabeled SFV at pH7.4 or pH 5.5 in the presence of 10 μM His-DIII and used trypsindigestion to quantitate the amount of retrieved E1 trimer (FIG. 11D).About 50% of the total E1 converted to a trypsin-resistant trimerconformation following the low pH pulse. The E1 population retrieved byeither E1a-1 or the antibody to the His-tag was strongly enriched intrypsin-resistant E1. Thus, domain III preferentially interacts with atrimeric conformation of E1, supporting a model in which the initialtrimerization of E1 produces a binding site for exogenous domain III.

DISCUSSION

We here demonstrate that exogenously added domain III can inhibit thealphavirus and flavivirus membrane fusion reactions. This is the firstdemonstration of such dominant-negative inhibition of the class IIfusion proteins. Studies of class I inhibition by dominant-negativepeptides such as T20 indicate that the speed of the fusion reactioncontrols its sensitivity to inhibition. Both the alphavirus andflavivirus fusion reactions are very rapidly triggered by low pH, withkinetics considerably faster than those of HIV-1, the target for T20.Nonetheless, domain III is able to inhibit the class II fusion reactionat the cell surface and within the normal endosomal entry pathway.Domain III inhibition thus provides proof of principle of adominant-negative inhibitor strategy for the class II fusion proteins,and demonstrates the key role of the class II trimer in virus fusion andinfection.

Our studies with SFV demonstrate that fusion is blocked at an early stepprior to lipid mixing, and that domain III stably interacts with atrimeric form of E1. Unlike the E1 monomer, this E1 trimer contains abinding site for domain III, presumably comprised of the domainIII/domain II “core trimer” with which domain III has been shown tointeract in the 3-D structure of the homotrimer (Gibbons et al, 2004b).Thus domain III inhibition identifies an important intermediate state inthe fusion reaction, which we interpret as reflecting formation of thecore E1 trimer prior to the folding-back of E1 domain III. Our resultsindicate that this foldback step is required for lipid mixing as well asfor full fusion. Given that the E1 fusion loop inserts in the targetmembrane prior to trimerization (Kielian et al, 1996), we assume thatthe domain III-sensitive state occurs after membrane insertion, butfurther studies will be needed to conclusively demonstrate this.

There are two populations of E1 homotrimers on the virus particlesduring fusion, HTs that interact with the target membrane and HTsoutside the fusion site that probably insert into the virus membrane.Interestingly, the concentrations of His-DIIIS required to inhibit SFVfusion in the FIA are lower than those that maximally inhibit HTformation under the same conditions. This suggests that a subset ofhomotrimers is the critical target for fusion inhibition, and that thisrelatively small number of trimers would initially be affected, prior todisruption of the bulk E1 trimers. We favor a model in which domain IIIis blocking the fold-back of critical E1 trimers at the fusion sitewhere the virus and target membranes are in close proximity. While ourdata do not rule out effects on E1 molecules outside the fusion zone,they do suggest that inhibition is unlikely to occur by completelyblocking bulk HT formation.

Studies of the membrane insertion of class II fusion protein ectodomainsindicate that insertion is highly cooperative (Gibbons et al, 2003,Stiasny et al, 2004). In the case of SFV, ectodomain insertion producesrings of 5-6 trimers, reflecting the physical associations of the fusionloops and domain III regions of adjacent HTs (Gibbons, 2000b; Gibbons etal., 2003). These cooperative interactions produce a volcano-likeassembly of E1 HTs that may help to induce membrane curvature at thefusion site. An alternative model for inhibition by domain III is thatit acts to inhibit such cooperative HT-HT interactions during fusion.While we hypothesize that these inter-trimer interactions are importantfor fusion, we feel that the strongest model for the action of domainIII is that it acts not to prevent interactions between adjacent HTs,but to inhibit the folding-back reaction within one E1 molecule. Thisagrees well with the ability of domain III to co-immunoprecipitate E1 inthe presence of octyl-glucoside, a detergent that we previously founddisrupted HT-HT interactions (Gibbons et al., 2003; Gibbons, 2004a). Italso agrees with the increase in inhibition and co-immunoprecipitationthat is observed when the stem is present on domain III, since no rolefor the stem in HT-HT interaction was observed in the previous studies.However, it is possible that domain III could be acting by somecombination of these two models. For example, prevention of E1 refoldingby binding of exogenous domain III could inhibit the ability of theviral domain III to interact with domain III on an adjacent trimer.

Given the speed of the SFV fusion reaction, it is perhaps surprisingthat exogenous domain III can compete with the endogenous domain III forbinding to the core HT. Such an inter-molecular interaction of domainIII would seem to be at a disadvantage compared with the intramolecularinteraction of the E1 domain III. Several factors may help to explainthis paradox. The movement of domain III in the full-length E1 may beconstrained by its attachment to the virus membrane through thestem/anchor domains. Indeed, we found that domain III binding to theectodomain trimer was not as efficient as binding to the full-lengthtrimer (data not shown), in keeping with the loss of the membrane anchorconstraint in the ectodomain. The structure of the E1 homotrimer alsoreveals that the linker region between domain I and domain III becomeshighly extended during the movement of domain III towards the fusionloop (Gibbons 2004B). This could provide an additional constraint to E1domain III movement, allowing the initial interaction of exogenousdomain III with the trimer. This initial binding of domain III could actto orient the stem region for its interaction with the core trimer. Inthis model, the domain III interaction would be the key first step ininhibition, followed by the close and sequential “zipping up” of thestem along the body of the trimer. This model also fits with thefindings that inhibition of the class I and SNARE fusion reactions ismost effective when it targets the initial membrane distal hairpininteraction.

While domain III is a useful basic research tool, its inhibitory actionhas important implications for the development of more clinically usefulinhibitors of the class II proteins. Inhibition by domain III wasobserved within a virus genus (SFV vs. Sindbis) but not between membersof the alphavirus genus and the flavivirus genus (SFV vs. DV2). Thisresult suggests that key amino acid contacts between domain III and thecore HT are conserved among viruses of the same genus. Simpleexamination of the HT structure and location of conserved residues doesnot clearly identify these critical target sites. However, sinceexogenous domain III showed stable binding to a trimeric E1 target, thisinteraction could be used as a general screen for peptides or smallmolecules that would block domain III-trimer binding. Given thecross-inhibition by domain III, such screens could have the potential toidentify broad-specificity small molecule inhibitors.

While the first class I inhibitors were peptides, more recently smallmolecules that target critical sites of interaction in the trimer ofhairpins have also been shown to act as fusion inhibitors (Clanci etal., 2004). Thus, although perhaps affecting only a subset of thecontacts involved in the domain III-core trimer interaction, smallmolecules could act as potent inhibitors of class II fusion with thepotential to act from within endocytic compartments.

Experimental Procedures

Cells and viruses. BHK-21 cells and C6/36 mosquito cells were culturedas previously described (Phalen and Kielian, 1991). SFV was awell-characterized plaque-purified isolate (Vashishtha et al, 1998), SINwas derived from the infectious clone of Toto 1101, VSV expressing GFP(VSV-GFP) was a generous gift from Dr. John K. Rose, DV2 (strain NewGuinea C) was kindly provided by Dr. Stacey L. Bartlett, and DV1 (strainWestern Pacific) was obtained from Dr. Richard Stockert. SFV, SIN andVSV-GFP were propagated in BHK-21 cells, while dengue viruses werepropagated in C6/36 cells in DME containing 2% heat-inactivated FCS and10 mM HEPES (pH 8.0).

Construction of domain III protein expression plasmids. DNA sequences ofSFV E1 domain III (with or without stem region) were amplified fromplasmid DG-1, and the DNA sequences of DV2 E domain III were obtained byRT-PCR on viral RNA extracted from DV2-infected C6/36 cells. Thesesequences were subcloned into protein expression plasmid pET-14b(Novogen) to express the exact domain III proteins with only one extrainitiator methionine, and were also subcloned into pRSET A plasmid(Invitrogen) to express N-terminal 6× Histidine tagged domain IIIproteins with extra N-terminal 36 amino acids (aa). The DNA fragmentssubcloned into expression vectors were confirmed by DNA sequencing(Genewiz). Sequences of several of the proteins described herein areprovided as SEQ ID NO:4-9.

Protein expression, refolding purification and concentration. Domain IIIproteins were expressed and refolded essentially as described in Zhanget al, 2002b. E. coli strain BL21 (DE3) competent cells (Novagen) weretransformed with domain III protein expression plasmids, and cultured inLuria-Bertani (LB) medium with 50 μg/ml carbenicillin until OD₆₀₀reached 0.6. Protein expression was then induced with 1 mMisopropylthiogalactosidase (IPTG) at 37° C. for 3 h. The cells wereharvested by centrifuge, resuspended in lysis buffer (10 mM Tris, 150 mMNaCl, 25% (w/v) sucrose, 1 mM EDTA and 10 mM DTT, pH 8.0), supplementedwith 0.1 mg/ml lysozyme, and frozen at −80° C. over night prior to bebroken up by sonication. The insoluble inclusion body (IB) was collectedby centrifuge at 20,000×g for 15 min, and washed 3 times with washingbuffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA and 10 mM DTT, pH 8.0) with0.5% Triton X-100 and then 2 times with the same washing buffer withoutdetergent. The washed inclusion body (IB) was solubilized in IB dilutionbuffer (6M guanidine-HCl, 10 mM NaAc and 5 mM EDTA, pH 4.6) with 1 mMDTT, and the protein concentration was determined by OD₂₈₀ usingtheoretically calculated extinction coefficient. In order to refold thedenatured protein, 16 mg IB in 16 ml IB dilution buffer was quicklydiluted into 1 L refolding buffer (100 mM Tris, 0.4 M arginine-HCl, 1 mMEDTA, 5 mM cysteamine, 0.5 mM cystamine and 0.5 mMphenylmethanesulfonylfluoride [PMSF], pH 8.5), and sat at 4° C. for 6-8h before the next IB addition. There are about 5 IB dilutions in eachprotein preparation. The refolded proteins were concentrated from 1 L to10 ml using an Amicon stirred ultrafiltration cell, loaded on SuperdexG-75 gel filtration column (Amersham Biosciences), and eluted withelution buffer (10 mM Tris, 50 mM NaCl, pH 8.0). The correctly refoldedprotein was separated from aggregates and collected prior toconcentration and buffer exchange to low salt buffer (10mM Tris, 100 μMNaCl, pH 7.4) by 5000 molecular weight cutoff Vivaspin concentrator(Vivascience).

The concentration of domain III proteins was determined by absorption at205 nm as described in Scopes, 1974. About 10 mg of dIII was producedfrom 100 mg of inclusion bodies purified from 1 liter of bacterialculture.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Protein samples were heated in SDS sample buffer (200 mM Tris, 4% SDS,10% glycerol, 0.02% bromophenol blue, pH 8.8) with or without 10 mM DTTat 95° C. or 30° C. for 3 min, and the alkylation was achieved byincubating with 30 mM iodoacetamide at 37° C. for 15 min. The processedprotein samples were then loaded and electrophoresed on SDS-acrylamidegel with standard Tris-glycine system, except in FIG. 6B where aTris-tricine system was used. ³⁵S-labeled proteins were quantified byPhosphorImager analysis with Image Quant version 1.2 software (MolecularDynamics, Sunnyvale, Calif.).

Mass spectrometry. Domain III proteins were measured by ESI massspectrometry on a ThermoFinnigan LCQ ion trap mass spectrometer.

Fusion infection assay (FIA). Fusion of virus with the plasma membraneof BHK cells was assayed in FIA as previous described (Vashishtha et al,1998). SFV, SIN and VSV-GFP were diluted in binding medium at pH 6.8(RPMI without bicarbonate plus 0.2% BSA and 10 mM HEPES[RPMI/BSA/HEPES]) supplemented with 20 mM MES and 20 mM NH₄Cl), whiledengue viruses were diluted in binding medium at pH 7.9 (RPMI/BSA/HEPESplus 20 mM NH₄Cl). BHK cells grown on 12-mm coverslips in 24-well platewere chilled on ice and washed twice with ice cold binding medium priorto binding with virus on ice for 90 to 120 min with gentle shaking(binding step). After virus binding, cells were washed 2 times withbinding medium to get rid of unbound virus. The virus-cell membranefusion was induced at 37° C. for 1 min in 200 μl pH medium(RPMI/BSA/HEPES plus 30 mM MES for pH 7.4 or RPMI/BSA/HEPES plus 30 mMsodium succinate for pH 6.0 or lower) (fusion step). Cells were put backon ice and washed 2 times with binding medium. SFV, SIN and VSV-GFPinfected BHK cells were incubated at 28° C. overnight in BHK growthmedium plus 20 mM NH₄Cl, while dengue viruses infected cells wereincubated in Medium S (minimum essential medium [MEM] supplemented with2% FCS) with 50 mM NH₄Cl for 3 h at 37° C. prior to growth at 37° C. for2 days in Medium S plus 20 mM NH₄Cl (culture step).

Compared to SFV and SIN, dengue viruses started fusion at higher pH (˜7)in FIA. DV2 and DV1 showed ˜50% fusion at pH 6.5, and reached maximalfusion when fusion pH was lowered to 6.2, where the fusion efficiency ofpre-bound dengue viruses was 30%.

Immunofluorescence microscopy. VSV-GFP infected cells were fixed in 3%formaldehyde at room temperature for 20 min, and VSV infected cellsexpressing GFP were observed directly without antibody staining underfluorescence microscopy. The cells infected by all other viruses werefixed in ice-cold methanol for 10 min. SFV and SIN infected cells werestained with rabbit polyclonal antisera against SFV or SIN, and thendetected by FITC-conjugated goat anti-rabbit antibody. DV2 and DV1infected cells were stained with mouse polyclonal hyperimmune ascitesfluid against DV2 (obtained from Dr. Robert B. Tesh), and then detectedby Alexa fluor 488 conjugated anti-mouse antibody (Molecular Probe). Foreach sample duplicate coverslips were evaluated at an infection levelof >200 cells/coverslip in the absence of inhibitor.

Assays for SFV E1 homotrimer formation with domain III proteins. Toassess the conformational change of SFV E1 protein during fusion in thepresence of domain III proteins, the 35S-labeled SFV was generated asdescribed before, and the fusion between ³⁵S-labeled SFV and BHK cellswere assayed as FIA. Immediately after the fusion step, the cells werelysed in lysis buffer (20 mM Tris, 100 mM NaCl, 1.5% octyl-glucoside, 1mM EDTA, pH 7.4 plus 1 μg/ml pepstatin, 50 μg/ml leupeptin, 0.1% BSA,100 μg/ml aprotitin and 1 mM PMSF). In order to assay the SDS resistantE1 homotrimer, one aliquot of lysates was added to SDS sample buffer andheated to 30° C. for 3 min prior to SDS-acrylamide 7% gel. Anotheraliquot of cell lysates was subjected to immunoprecipitation aspreviously described using different antibodies as indicated in FIG. 11,and the E1 protein bound to specific antibody was quantified afterresolved in SDS-PAGE on 11% gel.

To further test the trypsin resistance of the immunoprecipitated E1proteins, the immunoprecipitated pellets were resuspended in PBS with 1%TX-100, and were added to 1 mg/ml trypsin (freshly made in PBS/1%TX-100) to a final concentration of 125 μg/ml. The trypsin digestion wasat 37° C. for 1 h and then stopped by adding 5 mM PMSF. These mixtureswere added with SDS (final concentration as 2%), heated at 95° C. for 3min, and shaken vigorously at room temperature for 4 min, then repeatedtwice. The insoluble zysorbin was spun away, and the supernatants werecollected, precipitated in 1% TCA, washed in ice-cold acetone andsubjected to SDS-PAGE.

Pyrene-labeled SFV fusion with cell membrane. Pyrene-labeled SFV wasprepared as described before, and fused with BHK cell membraneessentially the same as FIA. In brief, BHK cells grown on 35 mm plateswere pre-bound with pyrene-labeled SFV (diluted in binding medium [pH6.8]) (MOI ˜2000) by incubation on ice for 120 min with gentle shaking.Unbound virus was washed away, and the virus fusion on plasma membranewas induced at 37° C. for 1 min in pH 7.4 or pH 5.5 medium with certainconcentrations of domain III proteins. Cells were put back on ice,washed once with binding medium, once with H—H solution (Hank's balancedsalt solution buffered with 10 mM HEPES, pH 7.4 and supplemented with 20mM NH₄Cl). The cells were then scraped off in H—H solution, transferredto a quartz cuvette, and pre-equilibrated in an AB-2 fluorometer for 1min prior to the fluorescence scanning at 37° C. The cells bound withpyrene-labeled SFV were excited at 343 nm, and the fluorescence emissionfrom 360 to 560 nm was recorded as average from 2 serial scanning. Thefluorescence signal from empty cells was subtracted from each sample.The monomer pyrene fluorescence peak (M) is at 397 nm, while theconcentrated pyrene probes originally in the viral membrane shows aneximer peak (Ex) at 475 nm. The lipid mixing between pyrene-labeled SFVand the target cells can be followed by the decrease of Ex/M ratio, i.e.the dilution of pyrene from viral membrane to cell plasma membrane.

Viral infection via regular endocytosis. SFV, SIN, VSV-GFP and DV2 werediluted in RPMI/BSA/HEPES (pH 7.2), mixed with various concentrations ofdomain III proteins, and seeded on BHK cells grown on 12-mm coverslipsin 24-well plates. The cells were kept in a 20° C. water bath for 1 h toallow the regular virus infection via endocytosis. The infection wasstopped by addition of growth medium plus NH₄Cl as in the culture stepof FIA.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

SEQ ID NO:s

A Domain III of a class II fusion protein of a Semliki Forest Virus SEQID NO: 1 VEAPTIIDLTCTVATCTHSSDFGGVLTLTYKTDKNGDCSVHSHSNVATLQEATAKVKTAGKVTLHFSTASASPSFVVSLCSARATCSASCEPP A stem region of a class II fusionprotein of a Semliki Forest Virus SEQ ID NO: 2KDHIVPYAASHSNVVFPDMSGTALSWVQK GenBank accession NP_819008: a class IIfusion protein from a Semliki Forest Virus (E protein). Domain III is inbold, the stem region is in italics, and the Domain I/Domain III linkeris underlined. SEQ ID NO: 3 1 yehstvmpnv vgfpykahie rpgyspltlqmqvvetslep tlnleyitce yktvvpspyv 61 kccgasecst kekpdyqckv ytgvypfmwggaycfcdsen tqlseayvdr sdvcrhdhas 121 aykahtaslk akvrvmygnv nqtvdvyvngdhavtiggtq fifgplssaw tpfdnkivvy 181 kdevfnqdfp pygsgqpgrf gdiqsrtvesndlyantalk larpspgmvh vpytqtpsgf 241 kywlkekgta lntkapfgcq iktnpvramncavgnipvsm nlpdsaftri veaptiidlt 301 ctvatcthss dfggvltlty ktnkngdcsvhshsnvatlq eatakvktag kvtlhfstas 361 aspsfvvslc saratcsasc epp kdhivpyaashsnvvfp dmsgtalswv qkisgglgaf 421 aigailvlvv vtciglrr SEQ ID NO: 4-7Domain III proteins derived from a Semliki Forest Virus E protein. SeeExample for nomenclature. DIII SEQ ID NO: 4MVEAPTIIDLTCTVATCTHSSDFGGVLTLTYKTDKNGDCSVHSHSNVATLQEATAKVKTAGKVTLHFSTASASPSFVVSLCSARATCSASCEPP (Met + 291-383) DIIIS SEQ ID NO: 5MVEAPTIIDLTCTVATCTHSSDFGGVLTLTYKTDKNGDCSVHSHSNVATLQEATAKVKTAGKVTLHFSTASASPSFVVSLCSARATCSASCEPPKDHIVPYAASHSNVVFPDMSGTALS WVQK (Met+ 291-412) His-DIII SEQ ID NO: 6MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSVEAPTIIDLTCTVATCTHSSDFGGVLTLTYKTDKNGDCSVHSHSNVATLQEATAKVKTAGKVTLHFSTASASPSFVVSLCSARATCSASCEPP (6-His tag underlined + 291-383) His-DIIIS SEQ ID NO: 7MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSVEAPTIIDLTCTVATCTHSSDFGGVLTLTYKTDKNGDCSVHSHSNVATLQEATAKVKTAGKVTLHFSTASASPSFVVSLCSARATCSASCEPPKDHIVPYAASHSNVVFPDMSGTALSWVQK (6-His tag underlined +291-412) SEQ ID NO: 8 and 9 Domain III proteins from Denaue virus 2 E.See Example for nomenclature. DV2DIIIH1 SEQ ID NO: 8MGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKRMAIL (Met+ 296-395 + H1 396-415) His-DV2DIII SEQ ID NO: 9MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKG (6-His tag underlined + 296-395) A Domain III of aclass II fusion protein of a Semliki Forest Virus with the DI/DIIIlinker SEQ ID NO: 10MNLPDSAFTRIVEAPTIIDLTCTVATCTHSSDFGGVLTLTYKTDKNGDCSVHSNVATLQEATAKVKTAGKVTLHFSTASASPSFVVSLCSARATCSASCEPP A DI/DIII linker from aclass II fusion protein of a Semliki Forest Virus SEQ ID NO: 11MNLPDSAFTRI

1. A method of inhibiting viral infection of a eukaryotic cell by atarget virus having a class II virus fusion protein, the methodcomprising combining the virus with an aqueous-soluble proteincomprising a domain equivalent to a Domain III of an Alphavirus class IIfusion protein, wherein the Domain III has SEQ ID NO:1.
 2. The method ofclaim 1, wherein the aqueous-soluble protein further comprises a regionequivalent to at least a portion of a stem region of an Alphavirus classII fusion protein, wherein the Alphavirus stem region has SEQ ID NO:2.3. The method of claim 1, wherein the aqueous-soluble protein furthercomprises a region equivalent to a DI/DIII linker region from anAlphavirus class II fusion protein, wherein the Alphavirus linker regionhas SEQ ID NO:11.
 4. The method of claim 1, wherein the aqueous-solubleprotein further comprises an oligohistidine moiety.
 5. The method ofclaim 1, wherein the aqueous-soluble protein comprises SEQ ID NO:1. 6.The method claim 1, wherein the aqueous-soluble protein is present at aconcentration of 50 nM or greater.
 7. The method of claim 1, wherein thetarget virus is a member of the Togaviridae, Flaviviridae, orBunyaviridae.
 8. The method of claim 1, wherein the target virus is anAlphavirus.
 9. The method of claim 8, wherein the Alphavirus is SemlikiForest virus, eastern equine encephalitis virus, western equineencephalitis virus, or Venezuelan equine encephalitis virus.
 10. Themethod of claim 1, wherein the target virus is a member of theFlaviviridae.
 11. The method of claim 10, wherein the target virus isdengue virus, hepatitis C virus, Japanese encephalitis virus, tick-borneencephalitis virus, yellow fever virus, West Nile virus, bovine viraldiarrhea virus, or swine fever virus.
 12. The method of claim 1, whereinthe target virus is a member of the Bunyaviridae.
 13. The method ofclaim 12, wherein the target virus is Crimean-Congo hemorrhagic fevervirus or tomato spotted wilt virus.
 14. The method of claim 1, whereinthe target virus is Semliki Forest virus or dengue virus.
 15. The methodof claim 1, wherein the aqueous-soluble protein is a portion of theclass II virus fusion protein of a virus in the same viral genus as thetarget virus.
 16. The method of claim 1, wherein the aqueous-solubleprotein is a portion of the class II virus fusion protein of the targetvirus.
 17. The method of claim 1, wherein the target virus is in aliving eukaryote.
 18. The method of claim 17, wherein the livingeukaryote is a mammal.
 19. A method of screening a test compound for theability to inhibit infection by a virus having a class II viral fusionprotein, the method comprising (a) combining the test compound with (i)an aqueous-soluble protein comprising a domain equivalent to a DomainIII of an Alphavirus fusion protein, wherein the Domain III has SEQ IDNO:1, and (ii) a core homotrimer of the class II viral fusion protein,then (b) determining whether the aqueous-soluble protein and the coretrimer are bound together, wherein reduced binding of the protein withthe core homotrimer in the presence of the test compound indicates thatthe test compound inhibits infection by the virus. 20-31. (canceled) 32.A method of screening a test compound for the ability to inhibitinfection by a virus having a class II viral fusion protein, the methodcomprising (a) combining the test compound with a class II viral fusionprotein, wherein the class II viral fusion protein comprises a Domain I,a Domain II, and a Domain III, and wherein the Domain III furthercomprises a fluorescent molecule that is quenched upon fold-back of theDomain III during trimerization, and (b) determining whether the testcompound reduces quenching of the fluorescent molecule upon conditionswhere quenching occurs in the absence of the test compound, whereinreduced quenching of the fluorescent molecule indicates that the testcompound inhibits infection by the virus.
 33. (canceled)
 34. Anaqueous-soluble protein comprising a portion of a class II viral fusionprotein, wherein the portion of the class II viral fusion proteincomprises (a) a domain equivalent to a Domain III of an Alphavirusfusion protein, wherein the Domain III has SEQ ID NO:1, and (b) a regionequivalent to at least a portion of a stem region of an Alphavirusfusion protein, wherein the stem region has SEQ ID NO:2. 35-44.(canceled)